Fused heterocyclic derivatives as phosphodiesterase inhibitors

ABSTRACT

Compounds of general structural formula (I) and use of the compounds and salts and solvates thereof, as therapeutic agents.

[0001] This invention relates to a series of compounds, to methods ofpreparing the compounds, to pharmaceutical compositions containing thecompounds, and to their use as therapeutic agents. In particular, theinvention relates to compounds that are potent and selective inhibitorsof cyclic guanosine 3′,5′-monophosphate specific phosphodiesterase(cGMP-specific PDE), in particular PDE5, and have utility in a varietyof therapeutic areas wherein such inhibition is considered beneficial,including the treatment of cardiovascular disorders and erectiledysfunction.

SUMMARY OF THE INVENTION

[0002] The present invention provides compounds of formula (I)

[0003] wherein R⁰, independently, is selected from the group consistingof halo and C₁₋₆alkyl;

[0004] R¹ is selected from the group consisting of hydrogen, C₁₋₆alkyl,C₂₋₆alkenyl, C₂₋₆alkynyl, haloC₁₋₆-alkyl, C₃₋₈cycloalkyl,C₃₋₈cycloalkylC₁₋₃alkyl, arylC₁₋₃-alkyl, and heteroarylC₁₋₃alkyl;

[0005] R² is selected from the group consisting of an optionallysubstituted monocyclic aromatic ring selected from the group consistingof benzene, thiophene, furan, and pyridine, and an optionallysubstituted bicyclic ring

[0006]  wherein the fused ring A is a 5- or 6-membered ring, saturatedor partially or fully unsaturated, and comprises carbon atoms andoptionally one or two heteroatoms selected from oxygen, sulfur, andnitrogen;

[0007] X is selected from the group consisting of NR^(a), O, S, CR³R⁴,CH₂CR³R⁵, and CR³R⁵CH₂;

[0008] R³ is selected from the group consisting of hydrogen andC₁₋₃alkyl;

[0009] or R¹ and R³ together represent a 3- or 4-membered alkyl oralkenyl chain component of a 5- or 6-membered ring,

[0010] or NR^(a), R¹, and the nitrogen to which R¹ is attached, form a5- or 6-membered heterocyclic ring;

[0011] R⁴ is selected from the group consisting of C₁₋₆alkyl,haloC₁₋₄alkyl, aryl, heteroaryl, Het, C₃₋₈-cycloalkyl, OR^(a),C(═O)OR^(a), C(═O)R^(a), C(═O)NR^(a)SO₂R^(b), C(═O)NR^(a)R^(b),C(═S)NR^(a)R^(b), C₁₋₆alkyleneC(═O)OR^(a), C₁₋₄alkyleneC(═O)NR^(a)R^(b),C₁₋₄alkyleneNR^(a)R^(b), C₁₋₄alkyleneOR^(a),C₁₋₄-alkyleneSO₂NR^(a)R^(b), C₁₋₄alkylenearyl, C₁₋₄alkyleneheteroaryl,C₁₋₄alkyleneHet, C₁₋₄alkyleneC(═O)C₁₋₄alkyleneHet, C₁₋₄alkyleneC(═O)Het,C₁₋₄alkyleneC(═O)OR^(a), C(═O)—C₁₋₄alkyleneHet,C(═O)NR^(a)alkyleneOR^(b), C(═O)NR^(a)C₁₋₄alkyleneHet,NR^(a)C₁₋₄alkyleneNR^(a)R^(b), NR^(a)C(═O)R^(b), NR^(a)C(═O)NR^(a)R^(b),N(SO₂C₁₋₄alkyl)₂, and NR^(a)(SO₂C₁₋₄alkyl), with the proviso that whenR³ is hydrogen, R⁴ is different from C₁₋₃alkyl;

[0012] or R¹ and R⁴ together represent a 3- or 4-membered carbocyclic orheterocyclic chain component, either saturated or unsaturated, of a 5-or 6-membered ring;

[0013] or R³ and R⁴ together represent a 4- to 6-membered alkyl oralkenyl chain component of a 5- to 7-membered ring;

[0014] R⁵ is selected from the group consisting of hydrogen, C₁₋₆alkyl,haloC₁₋₄alkyl, aryl, heteroaryl, Het, C₃₋₈cycloalkyl, OR^(a),C(═O)OR^(a), C(═O)R^(a), C(═O)—NR^(a)SO₂R^(b), C(═O)NR^(a)R^(b),C(═S)NR^(a)R^(b), C₁₋₆alkyleneC(═O)OR^(a), C₁₋₄alkyleneC(═O)NR^(a)R^(b),C₁₋₄alkyleneNR^(a)R^(b), C₁₋₄alkyleneOR^(a), C₁₋₄alkyleneSO₂NR^(a)R^(b),C₁₋₄alkylenearyl, C₁₋₄alkyleneheteroaryl, C₁₋₄alkyleneHet,C₁₋₄alkyleneC(═O)C₁₋₄-alkyleneHet, C₁₋₄alkyleneC(═O)Het,C₁₋₄alkyleneC(═O)—OR^(a), C(═O)C₁₋₄alkyleneHet,C(═O)NR^(a)alkyleneOR^(b), C(═O)—NR^(a)C₁₋₄alkyleneHet,NR^(a)C₁₋₄alkyleneNR^(a)R^(b), NR^(a)C(═O)R^(b), NR^(a)C(═O)NR^(a)R^(b),N(SO₂C₁₋₄alkyl)₂, and NR^(a)(SO₂C₁₋₄alkyl);

[0015] R^(a) and R^(b), independently, are selected from the groupconsisting of hydrogen, C₁₋₈alkyl, aryl, heteroaryl, haloC₁₋₆alkyl,C₁₋₄alkyleneHet, arylC₁₋₃-alkyl, heteroarylC₁₋₃alkyl, C₁₋₄alkylenearyl,C₃₋₈-cycloalkyl, C₁₋₃alkyleneheteroaryl, and Het;

[0016] Het is a 4- to 7-membered heterocyclic group, saturated orpartially unsaturated, containing at least one heteroatom selected fromthe group consisting of oxygen, nitrogen, and sulfur, and optionallysubstituted with one or more of C₁₋₄alkyl, NR^(a)R^(b), and C(═O)OR^(a);

[0017] q is 0, 1, 2, 3, or 4; and

[0018] pharmaceutically acceptable salts and solvates (e.g., hydrates)thereof.

[0019] As used herein, the term “alkyl” includes straight chained andbranched hydrocarbon groups containing the indicated number of carbonatoms, typically methyl, ethyl, and straight chain and branched propyland butyl groups. The hydrocarbon group can contain up to 16 carbonatoms. The term “alkyl” includes “bridged alkyl,” i.e., a C₆-C₁₆bicyclic or polycyclic hydrocarbon group, for example, norbornyl,adamantyl, bicyclo[2.2.2]octyl, bicyclo[2.2.1]heptyl,bicyclo[3.2.1]octyl, or decahydronaphthyl. The term “cycloalkyl” isdefined as a cyclic C₃-C₈ hydrocarbon group, cyclopropyl, cyclobutyl,cyclohexyl, and cyclopentyl. The terms “alkenyl” and “alkynyl” aredefined similarly to the term “alkyl,” except the hydrocarbon groupcontains a carbon-carbon double bond or carbon-carbon triple bond,respectively.

[0020] The term “alkylene” refers to an alkyl group having asubstituent. For example, the term “C₁₋₃alkylenearyl” refers to an alkylgroup containing one to three carbon atoms and substituted with an arylgroup. The term “alkenylene” as used herein is similarly defined, andcontains the indicated number of carbon atoms and a carbon-carbon doublebond, and includes straight chained and branched alkenylene groups, likeethyenylene.

[0021] The term “halo” or “halogen” is defined herein to includefluorine, bromine, chlorine, and iodine.

[0022] The term “haloalkyl” is defined herein as an alkyl groupsubstituted with one or more halo substituents, either fluoro, chloro,bromo, iodo, or combinations thereof. Similarly, “halocycloalkyl” isdefined as a cycloalkyl group having one or more halo substituents.

[0023] The term “aryl,” alone or in combination, is defined herein as amonocyclic or polycyclic aromatic group, preferably a monocyclic orbicyclic aromatic group, e.g., phenyl or naphthyl, that can beunsubstituted or substituted, for example, with one or more, and inparticular one to three, hydroxy, hydroxyalkyl, alkoxy, alkoxyalkyl,haloalkyl, nitro, amino, carboxy (CO₂H), CO₂R^(a), alkylamino,acylamino, alkylthio, alkylsulfinyl, and alkylsulfonyl. Exemplary arylgroups include phenyl, naphthyl, tetrahydronaphthyl, 2-chlorophenyl,3-chlorophenyl, 4-chlorophenyl, 2-methylphenyl, 4-methoxyphenyl,3-trifluoromethylphenyl, 4-nitrophenyl, and the like. The terms“arylC₁₋₃alkyl” and “heteroarylC₁₋₃alkyl” are defined as an aryl orheteroaryl group having a C₁₋₃alkyl substituent.

[0024] The term “heteroaryl” is defined herein as a monocyclic orbicyclic ring system containing one or two aromatic rings and containingat least one nitrogen, oxygen, or sulfur atom in an aromatic ring, andwhich can be unsubstituted or substituted, for example, with one ormore, and in particular one to three, substituents, like halo, alkyl,hydroxy, hydroxyalkyl, alkoxy, alkoxyalkyl, haloalkyl, nitro, amino,carboxy (CO₂H), CO₂R^(a), alkylamino, acylamino, alkylthio,alkylsulfinyl, and alkylsulfonyl. Nonlimiting examples of heteroarylgroups include thienyl, furyl, pyridyl, oxazolyl, quinolyl, isoquinolyl,indolyl, triazolyl, tetrazolyl, isothiazolyl, isoxazolyl, imidizolyl,benzothiazolyl, pyrazinyl, pyrimidinyl, thiazolyl, and thiadiazolyl.

[0025] The term “Het” includes a 4- to 7-membered heterocycloalkylgroup, including, but not limited to, morpholinyl, piperidyl,pyrrolidinyl, or piperazinyl.

[0026] The term “alkoxy” is defined as —OR, wherein R is alkyl.

[0027] The term “alkoxyalkyl” is defined as an alkyl group wherein ahydrogen has been replaced by an alkoxy group. The term“(alkylthio)alkyl” is defined similarly, except a sulfur atom, ratherthan an oxygen atom, is present.

[0028] The term “hydroxy” is defined as —OH.

[0029] The term “hydroxyalkyl”is defined as a hydroxy group appended toan alkyl group.

[0030] The term “amino” is defined as —NH₂, and the term “alkylamino” isdefined as —NR₂, wherein at least one R is alkyl and the second R isalkyl or hydrogen.

[0031] The term “acylamino” is defined as RC(═O)N, wherein R is alkyl oraryl.

[0032] The term “alkylthio” is defined as —SR, where R is alkyl.

[0033] The term “alkylsulfinyl” is defined as R—SO₂, where R is alkyl.

[0034] The term “alkylsulfonyl” is defined as R—SO₃, where R is alkyl.

[0035] The term “nitro” is defined as —NO₂.

[0036] The term “trifluoromethyl” is defined as —CF₃.

[0037] The term “trifluoromethoxy” is defined as —OCF₃.

[0038] The term “cyano” is defined as —CN.

[0039] The term “spiro” as used herein refers to a group having twocarbon atoms directly bonded to a carbon atom of the tetracyclic ringsystem.

[0040] In preferred embodiments, R⁴ and R⁵ are selected from the groupconsisting of C₁₋₆alkyl, C₁₋₆alkyleneC(═O)OR^(a), C(═O)OR^(a),C(═S)NR^(a)R^(b), C₅₋₇spiro when combined with R³,C₁₋₄alkyleneC(═O)NR^(a)R^(b); C₁₋₄-alkyleneNR^(a)R^(b),C₁₋₄alkyleneOR^(a), C₁₋₄alkyleneSO₂NR^(a)R^(b), C₁₋₄alkyleneheteroaryl,haloC₁₋₄alkyl, C₁₋₄alkyleneHet, C₁₋₄alkyleneC(═O)C₁₋₄alkyleneHet, andC₁₋₄alkylenearyl, and R^(a) and R^(b), independently, are selected fromthe group consisting of C₁₋₈alkyl, hydrogen, C₁₋₄alkylenearyl, Het,C₁₋₄alkyleneHet, C₃₋₈cycloalkyl, and haloC₁₋₆alkyl. Het, aryl, andheteroaryl can be substituted with one or more of C(═O)OR^(a),NR^(a)R^(b), and C₁₋₆alkyl.

[0041] In a more preferred group of compounds of formula (I), X isrepresented by CH₂CH₂, NH, or NCH₃; R³ is hydrogen or methyl; and R⁴ isselected from the group consisting of C₁₋₄alkyl, C₁₋₄alkyleneC(═O)NH₂,C₁₋₄alkyleneNH₂, C₁₋₄alkyleneC(═O)OC₁₋₇alkyl, C₁₋₆alkyleneC(═O)OR^(a),C₁₋₃alkyleneOH, C₁₋₄alkyleneSO₂NH₂, C₁₋₄alkyleneOC₁₋₃alkylenearyl,haloC₁₋₃alkyl, C₁₋₃alkylenearyl, C₁₋₄alkyleneC(═O)OC₁₋₃haloalkyl,

[0042] In another preferred embodiment, R³ and R⁴ are taken together toform a 5- or 6-membered spiro group.

[0043] A preferred R² group is represented by

[0044] The bicyclic ring

[0045] can represent, for example, naphthalene or indene, or aheterocyclic, such as benzoxazole, benzothiazole, benzisoxazole,benzimidazole, quinoline, indole, benzothiophene, or benzofuran, or

[0046] wherein n is an integer 1 or 2, and Y and Z, independently,represent CH₂, O, S, or NR^(a). In a preferred group of compounds offormula (I), R² is represented by an optionally substituted bicyclicring

[0047] wherein n is 1 or 2, and Y and Z are each CH₂ or O. Especiallypreferred R² bicyclic rings include

[0048] Within this particular group of compounds, nonlimiting examplesof substituents are halogen (e.g., chlorine), hydroxy, C₁₋₃alkyl (e.g.,methyl, ethyl, or i-propyl), C₁₋₃alkoxy (e.g., methoxy or ethoxy),CO₂R^(b), halomethyl (e.g., trifluoromethoxy), cyano, nitro, andNR^(a)R^(b).

[0049] An especially preferred subclass of compounds within the generalscope of formula (I) is represented by compounds of formula (II)

[0050] and pharmaceutically acceptable salts and solvates (e.g.,hydrates) thereof.

[0051] Compounds of formula (I) can contain one or more asymmetriccenter, and, therefore, can exist as stereoisomers. The presentinvention includes both mixtures and separate individual stereoisomersof the compounds of formula (I). Compounds of formula (I) also can existin tautomeric forms, and the invention includes both mixtures andseparate individual tautomers thereof.

[0052] Pharmaceutically acceptable salts of the compounds of formula (I)can be acid addition salts formed with pharmaceutically acceptableacids. Examples of suitable salts include, but are not limited to, thehydrochloride, hydrobromide, sulfate, bisulfate, phosphate, hydrogenphosphate, acetate, benzoate, succinate, fumarate, maleate, lactate,citrate, tartrate, gluconate, methanesulfonate, benzenesulfonate, andp-toluenesulfonate salts. The compounds of the formula (I) also canprovide pharmaceutically acceptable metal salts, in particular alkalimetal salts and alkaline earth metal salts, with bases. Examples includethe sodium, potassium, magnesium, and calcium salts.

[0053] Compounds of the present invention are potent and selectiveinhibitors of cGMP-specific PDE5. Thus, compounds of formula (I) are ofinterest for use in therapy, specifically for the treatment of a varietyof conditions where selective inhibition of PDE5 is considered to bebeneficial.

[0054] Phosphodiesterases (PDEs) catalyze the hydrolysis of cyclicnucleotides, such as cyclic adenosine monophosphate (cAMP) and cyclicguanosine monophosphate (cGMP). The PDEs have been classified into atleast seven isoenzyme families and are present in many tissues (J. A.Beavo, Physiol. Rev., 75, p. 725 (1995)).

[0055] PDE5 inhibition is a particularly attractive target. A potent andselective inhibitor of PDE5 provides vasodilating, relaxing, anddiuretic effects, all of which are beneficial in the treatment ofvarious disease states. Research in this area has led to several classesof inhibitors based on the cGMP basic structure (E. Sybertz et al.,Expert. Opin. Ther. Pat., 7, p. 631 (1997)).

[0056] The biochemical, physiological, and clinical effects of PDE5inhibitors therefore suggest their utility in a variety of diseasestates in which modulation of smooth muscle, renal, hemostatic,inflammatory, and/or endocrine function is desirable. The compounds offormula (I), therefore, have utility in the treatment of a number ofdisorders, including stable, unstable, and variant (Prinzmetal) angina,hypertension, pulmonary hypertension, congestive heart failure, acuterespiratory distress syndrome, acute and chronic renal failure,atherosclerosis, conditions of reduced blood vessel patency (e.g.,postpercutaneous transluminal coronary or carotid angioplasty, orpost-bypass surgery graft stenosis), peripheral vascular disease,vascular disorders, such as Raynaud's disease, thrombocythemia,inflammatory diseases, stroke, bronchitis, chronic asthma, allergicasthma, allergic rhinitis, glaucoma, osteoporosis, preterm labor, benignprostatic hypertrophy, peptic ulcer, male erectile dysfunction, femalesexual dysfunction, and diseases characterized by disorders of gutmotility (e.g., irritable bowel syndrome).

[0057] An especially important use is the treatment of male erectiledysfunction, which is one form of impotence and is a common medicalproblem. Impotence can be defined as a lack of power, in the male, tocopulate, and can involve an inability to achieve penile erection orejaculation, or both. The incidence of erectile dysfunction increaseswith age, with about 50% of men over the age of 40 suffering from somedegree of erectile dysfunction.

[0058] In addition, a further important use is the treatment of femalearousal disorder. Female arousal disorders are defined as a recurrentinability to attain or maintain an adequate lubrication/swellingresponse of sexual excitement until completion of sexual activity. Thearousal response consists of vasocongestion in the pelvis, vaginallubrication, and expansion and swelling of external genitalia.

[0059] It is envisioned, therefore, that compounds of formula (I) areuseful in the treatment of male erectile dysfunction and female arousaldisorder. Thus, the present invention concerns the use of compounds offormula (I), or a pharmaceutically acceptable salt thereof, or apharmaceutical composition containing either entity, for the manufactureof a medicament for the curative or prophylactic treatment of erectiledysfunction in a male animal and arousal disorder in a female animal,including humans.

[0060] The term “treatment” includes preventing, lowering, stopping, orreversing the progression or severity of the condition or symptoms beingtreated. As such, the term “treatment” includes both medical therapeuticand/or prophylactic administration, as appropriate.

[0061] It also is understood that “a compound of formula (I),” or aphysiologically acceptable salt or solvate thereof, can be administeredas the neat compound, or as a pharmaceutical composition containingeither entity.

[0062] Although the compounds of the invention are envisioned primarilyfor the treatment of sexual dysfunction in humans, such as male erectiledysfunction and female arousal disorder, they also can be used for thetreatment of other disease states.

[0063] A further aspect of the present invention, therefore, isproviding a compound of formula (I) for use in the treatment of stable,unstable, and variant (Prinzmetal) angina, hypertension, pulmonaryhypertension, chronic obstructive pulmonary disease, congestive heartfailure, acute respiratory distress syndrome, acute and chronic renalfailure, atherosclerosis, conditions of reduced blood vessel patency(e.g., post-PTCA or post-bypass graft stenosis), peripheral vasculardisease, vascular disorders such as Raynaud's disease, thrombocythemia,inflammatory diseases, prophylaxis of myocardial infarction, prophylaxisof stroke, stroke, bronchitis, chronic asthma, allergic asthma, allergicrhinitis, glaucoma, osteoporosis, preterm labor, benign prostatichypertrophy, male and female erectile dysfunction, or diseasescharacterized by disorders of gut motility (e.g., IBS).

[0064] According to another aspect of the present invention, there isprovided the use of a compound of formula (I) for the manufacture of amedicament for the treatment of the above-noted conditions anddisorders.

[0065] In a further aspect, the present invention provides a method oftreating the above-noted conditions and disorders in a human or nonhumananimal body which comprises administering to said body a therapeuticallyeffective amount of a compound of formula (I).

[0066] Compounds of the invention can be administered by any suitableroute, for example by oral, buccal, inhalation, sublingual, rectal,vaginal, transurethral, nasal, topical, percutaneous, i.e., transdermal,or parenteral (including intravenous, intramuscular, subcutaneous, andintracoronary) administration. Parenteral administration can beaccomplished using a needle and syringe, or using a high pressuretechnique, like POWDERJECT™.

[0067] Oral administration of a compound of the invention is thepreferred route. Oral administration is the most convenient and avoidsthe disadvantages associated with other routes of administration. Forpatients suffering from a swallowing disorder or from impairment of drugabsorption after oral administration, the drug can be administeredparenterally, e.g., sublingually or buccally.

[0068] Compounds and pharmaceutical compositions suitable for use in thepresent invention include those wherein the active ingredient isadministered in an effective amount to achieve its intended purpose.More specifically, a “therapeutically effective amount” means an amounteffective to prevent development of, or to alleviate the existingsymptoms of, the subject being treated. Determination of the effectiveamounts is well within the capability of those skilled in the art,especially in light of the detailed disclosure provided herein.

[0069] A “therapeutically effective dose” refers to that amount of thecompound that results in achieving the desired effect. Toxicity andtherapeutic efficacy of such compounds can be determined by standardpharmaceutical procedures in cell cultures or experimental animals,e.g., for determining the LD₅₀ (the dose lethal to 50% of thepopulation) and the ED₅₀ (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index, which is expressed as the ratio between LD₅₀ andED₅₀. Compounds which exhibit high therapeutic indices are preferred.The data obtained from such data can be used in formulating a dosagerange for use in humans. The dosage of such compounds preferably lieswithin a range of circulating concentrations that include the ED₅₀ withlittle or no toxicity. The dosage can vary within this range dependingupon the dosage form employed, and the route of administration utilized.

[0070] The exact formulation, route of administration, and dosage can bechosen by the individual physician in view of the patient's condition.Dosage amount and interval can be adjusted individually to provideplasma levels of the active moiety which are sufficient to maintain thetherapeutic effects.

[0071] The amount of composition administered is dependent on thesubject being treated, on the subject's weight, the severity of theaffliction, the manner of administration, and the judgment of theprescribing physician.

[0072] Specifically, for administration to a human in the curative orprophylactic treatment of the conditions and disorders identified above,oral dosages of a compound of formula (I) generally are about 0.5 toabout 1000 mg daily for an average adult patient (70 kg). Thus, for atypical adult patient, individual tablets or capsules contain 0.2 to 500mg of active compound, in a suitable pharmaceutically acceptable vehicleor carrier, for administration in single or multiple doses, once orseveral times per day. Dosages for intravenous, buccal, or sublingualadministration typically are 0.1 to 500 mg per single dose as required.In practice, the physician determines the actual dosing regimen which ismost suitable for an individual patient, and the dosage varies with theage, weight, and response of the particular patient. The above dosagesare exemplary of the average case, but there can be individual instancesin which higher or lower dosages are merited, and such are within thescope of this invention.

[0073] For human use, a compound of the formula (I) can be administeredalone, but generally is administered in admixture with a pharmaceuticalcarrier selected with regard to the intended route of administration andstandard pharmaceutical practice. Pharmaceutical compositions for use inaccordance with the present invention thus can be formulated in aconventional manner using one or more physiologically acceptablecarriers comprising excipients and auxiliaries that facilitateprocessing of compounds of formula (I) into preparations which can beused pharmaceutically.

[0074] These pharmaceutical compositions can be manufactured in aconventional manner, e.g., by conventional mixing, dissolving,granulating, dragee-making, levigating, emulsifying, encapsulating,entrapping, or lyophilizing processes. Proper formulation is dependentupon the route of administration chosen. When a therapeuticallyeffective amount of a compound of the present invention is administeredorally, the composition typically is in the form of a tablet, capsule,powder, solution, or elixir. When administered in tablet form, thecomposition can additionally contain a solid carrier, such as a gelatinor an adjuvant. The tablet, capsule, and powder contain about 5 to about95% compound of the present invention, and preferably from about 25 toabout 90% compound of the present invention. When administered in liquidform, a liquid carrier such as water, petroleum, or oils of animal orplant origin can be added. The liquid form of the composition canfurther contain physiological saline solution, dextrose or othersaccharide solutions, or glycols. When administered in liquid form, thecomposition contains about 0.5 to about 90% by weight of a compound ofthe present invention, and preferably about 1 to about 50% of a compoundof the present invention.

[0075] When a therapeutically effective amount of a compound of thepresent invention is administered by intravenous, cutaneous, orsubcutaneous injection, the composition is in the form of apyrogen-free, parenterally acceptable aqueous solution. The preparationof such parenterally acceptable solutions, having due regard to pH,isotonicity, stability, and the like, is within the skill in the art. Apreferred composition for intravenous, cutaneous, or subcutaneousinjection typically contains, in addition to a compound of the presentinvention, an isotonic vehicle.

[0076] For oral administration, the compounds can be formulated readilyby combining a compound of formula (I) with pharmaceutically acceptablecarriers well known in the art. Such carriers enable the presentcompounds to be formulated as tablets, pills, dragees, capsules,liquids, gels, syrups, slurries, suspensions and the like, for oralingestion by a patient to be treated. Pharmaceutical preparations fororal use can be obtained by adding a compound of formula (I) with asolid excipient, optionally grinding a resulting mixture, and processingthe mixture of granules, after adding suitable auxiliaries, if desired,to obtain tablets or dragee cores. Suitable excipients include, forexample, fillers and cellulose preparations. If desired, disintegratingagents can be added.

[0077] For administration by inhalation, compounds of the presentinvention are conveniently delivered in the form of an aerosol spraypresentation from pressurized packs or a nebulizer, with the use of asuitable propellant. In the case of a pressurized aerosol, the dosageunit can be determined by providing a valve to deliver a metered amount.Capsules and cartridges of, e.g., gelatin, for use in an inhaler orinsufflation can be formulated containing a powder mix of the compoundand a suitable powder base such as lactose or starch.

[0078] The compounds can be formulated for parenteral administration byinjection, e.g., by bolus injection or continuous infusion. Formulationsfor injection can be presented in unit dosage form, e.g., in ampules orin multidose containers, with an added preservative. The compositionscan take such forms as suspensions, solutions, or emulsions in oily oraqueous vehicles, and can contain formulatory agents such as suspending,stabilizing, and/or dispersing agents.

[0079] Pharmaceutical formulations for parenteral administration includeaqueous solutions of the active compounds in water-soluble form.Additionally, suspensions of the active compounds can be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils or synthetic fatty acid esters. Aqueousinjection suspensions can contain substances which increase theviscosity of the suspension. Optionally, the suspension also can containsuitable stabilizers or agents that increase the solubility of thecompounds and allow for the preparation of highly concentratedsolutions. Alternatively, a present composition can be in powder formfor constitution with a suitable vehicle, e.g., sterile pyrogen-freewater, before use.

[0080] Compounds of the present invention also can be formulated inrectal compositions, such as suppositories or retention enemas, e.g.,containing conventional suppository bases. In addition to theformulations described previously, the compounds also can be formulatedas a depot preparation. Such long-acting formulations can beadministered by implantation (for example, subcutaneously orintramuscularly) or by intramuscular injection. Thus, for example, thecompounds can be formulated with suitable polymeric or hydrophobicmaterials (for example, as an emulsion in an acceptable oil) or ionexchange resins, or as sparingly soluble derivatives, for example, as asparingly soluble salt.

[0081] Many of the compounds of the present invention can be provided assalts with pharmaceutically compatible counterions. Suchpharmaceutically acceptable base addition salts are those salts thatretain the biological effectiveness and properties of the free acids,and that are obtained by reaction with suitable inorganic or organicbases.

[0082] In particular, a compound of formula (I) can be administeredorally, buccally, or sublingually in the form of tablets containingexcipients, such as starch or lactose, or in capsules or ovules, eitheralone or in admixture with excipients, or in the form of elixirs orsuspensions containing flavoring or coloring agents. Such liquidpreparations can be prepared with pharmaceutically acceptable additives,such as suspending agents. A compound also can be injected parenterally,for example, intravenously, intramuscularly, subcutaneously, orintracoronarily. For parenteral administration, the compound is bestused in the form of a sterile aqueous solution which can contain othersubstances, for example, salts, or monosaccharides, such as mannitol orglucose, to make the solution isotonic with blood.

[0083] For veterinary use, a compound of formula (I) or a nontoxic saltthereof, is administered as a suitably acceptable formulation inaccordance with normal veterinary practice. The veterinarian can readilydetermine the dosing regimen and route of administration that is mostappropriate for a particular animal.

[0084] Thus, the invention provides in a further aspect a pharmaceuticalcomposition comprising a compound of the formula (I), together with apharmaceutically acceptable diluent or carrier therefor. There isfurther provided by the present invention a process of preparing apharmaceutical composition comprising a compound of formula (I), whichprocess comprises mixing a compound of formula (I), together with apharmaceutically acceptable diluent or carrier therefor.

[0085] In a particular embodiment, the invention includes apharmaceutical composition for the curative or prophylactic treatment oferectile dysfunction in a male animal, or arousal disorder in a femaleanimal, including humans, comprising a compound of formula (I) or apharmaceutically acceptable salt thereof, together with apharmaceutically acceptable diluent or carrier.

[0086] Compounds of formula (I) can be prepared by any suitable methodknown in the art, or by the following processes which form part of thepresent invention. In the methods below, R⁰ through R⁵, R^(a), R^(b),and X are as defined in structural formula (I) above. In particular,Daugan U.S. Pat. No. 5,859,006, incorporated herein by reference,discloses preparation of a compound of structural formula (III).

[0087] In short, the compound of structural formula (III), i.e., thecis-isomer of Intermediates 1 and 2 of Daugan U.S. Pat. No. 5,859,006was prepared according to the following reaction scheme:

[0088] A compound of structural formula (I) is prepared similarly byreacting a tryptophan ester, or a tryptophan ester substituted withsuitable R⁰ substituents, with a suitable aldehyde to provide thedesired R² substituent. The resulting product then is cyclized byreaction with a suitable amine to provide a compound of structuralformula (I). The cyclization reaction is disclosed in Daugan U.S. Pat.No. 5,859,006.

[0089] In the synthesis of compounds of structural formula (I),protecting compounds and protecting groups, like benzyl chloroformateand trichloroethyl chloroformate, which are well known to personsskilled in the art, can be used. Such protecting groups are disclosed,for example, in T. W. Greene et al. “Protective Groups in OrganicSynthesis, Third Edition,” John Wiley and Sons, Inc., NY, N.Y. (1999).These protecting groups are removed in the final steps of the synthesisunder basic, acidic, or hydrogenolytic conditions which are readilyapparent to those skilled in the art. By employing appropriatemanipulation and protection of chemical functionalities, synthesis ofcompounds of structural formula (I) not specifically set forth hereincan be accomplished by methods analogous to the schemes set forth below.For example, the structure of a compound of structural formula (I) canbe varied by using an appropriate aldehyde to change the identity of R²,or by using a halo or alkyl phenyl-substituted tryptophan ester.

[0090] Compounds of formula (I) can be converted to other compounds offormula (I). Thus, for example, when a compound contains a substitutedaromatic ring, it is possible to prepare another suitably substitutedcompound of formula (I). Examples of appropriate interconversionsinclude, but are not limited to, OR^(a) to hydroxy by suitable means(e.g., using a agent such as SnCl₂ or a palladium catalyst, such aspalladium-on-carbon), or amino to substituted amino, such as alkylamine,using standard acylating or sulfonylating conditions.

[0091] Compounds of structural formula (I) can be made accordingly toone of the following four nonlimiting synthetic routes.

[0092] Method A

[0093] The 1,2,3,4-tetrahydro-β-carbolines of general formula (IV) canbe prepared by the Pictet-Spengler reaction as set forth in Daugan U.S.Pat. No. 5,859,006 and in A. Madrigal et al., J. Org. Chem., 63, page2724 (1998), for example. The resulting secondary amine then is treatedwith either an amino acid or an acid halide, under suitable acylationconditions, to form an amide-ester. Ring cyclization to form thediketopiperazine (I) is accomplished by intramolecular amine attack onthe ester. The amine can be derived from a suitable side chain bearing aleaving group that reacts with primary amine compound (V).

[0094] Method B

[0095] The diketopiperazine (I) also can be prepared by reaction of atryptophan with an amino acid under typical peptide coupling conditionsto form an N-acyltryptophan. Ring cyclization to form diketopiperazine(I) is accomplished by intramolecular amine attack on the ester. Theresulting piperazine can undergo a condensation with an aldehyde undermodified Pictet-Spengler conditions (see T. A. Miller et al., Bioorg.Med. Chem. Lett., 8, p. 1065 (1998), for example) to give theβ-carboline.

[0096] Method C

[0097] The β-carboline skeleton also can be prepared using theBischler-Napieralski reaction, which is a cyclodehydration of anacylated tryptophan, as disclosed in W. M. Whaley et al., Org. React.,VI, pp. 74-150 (1951). P₂O₅ and POCl₃ are the most common cyclizationreagents. Reduction of the imine with NaBH₄, for example, yields the1,2,3,4-tetrahydro-β-carboline.

[0098] A modified method, which avoids the potential for racemisation,includes acylating the amine of tryptophan first, followed by conversionto the thioamide with, for example, with Lawesson's reagent. Treatmentof the thioamide with an alkyl halide or acyl halide provides theiminium halide. Reduction of the crude iminium halide with sodiumborohydride (NaBH₄) at reduced temperature stereo-selectively providesthe 1,2,3,4-tetrahydro-β-carboline.

[0099] Method D

[0100] In addition, the β-carboline can be treated with a chloroformateto form a 2,3-dicarboxylic acid diester. Treatment of the diester with,for example, a hydrazine, provides compounds of general formula (I) whenX contains nitrogen (see K. Winterfield et al., Arch. Pharmaz, 304, 216(1971) and E. H. White et al., J. Org. Chem., 32, 1921 (1967).

[0101] Compounds of formula (I) can be prepared by the methods above asindividual stereoisomers from the appropriate stereoisomer of formula(III) or as a racemic mixture from the appropriate racemic compound offormula (III). Individual stereoisomers of the compounds of theinvention can be prepared from racemates by resolution using methodsknown in the art for the separation of racemic mixtures into theirconstituent stereoisomers, for example, using HPLC on a chiral column,such as Hypersil naphthyl urea, or using separation of salts ofstereoisomers. Compounds of the invention can be isolated in associationwith solvent molecules by crystallization from, or evaporation of, anappropriate solvent.

[0102] The pharmaceutically acceptable acid addition salts of thecompounds of formula (I) that contain a basic center can be prepared ina conventional manner. For example, a solution of the free base can betreated with a suitable acid, either neat or in a suitable solution, andthe resulting salt isolated either by filtration or by evaporation undervacuum of the reaction solvent. Pharmaceutically acceptable baseaddition salts can be obtained in an analogous manner by treating asolution of a compound of formula (I) with a suitable base. Both typesof salt can be formed or interconverted using ion-exchange resintechniques. Thus, according to a further aspect of the invention, amethod for preparing a compound of formula (I) or a salt or solvate(e.g., hydrate) is provided, followed by (i) salt formation, or (ii)solvate (e.g., hydrate) formation.

[0103] The following abbreviations are used hereafter in theaccompanying examples: rt (room temperature), min (minute), h (hour), g(gram), mmol (millimole), m.p. (melting point), eq (equivalents), quant(quantitative), L (liter), mL (milliliter), μL (microliters), DCC(dicyclohexylcarbodiimide), Cbz (benzylcarbamate), DMSO (dimethylsulfoxide), CH₂Cl₂ (dichloromethane), CHCl₃ (chloroform), IPA (isopropylalcohol), TFA (trifluoroacetic acid), EtOH (ethanol), AcOH (aceticacid), EtOAc (ethyl acetate), MeOH (methanol), Et₃N (triethylamine), DMF(dimethylformamide), EtOAc (ethyl acetate), and THF (tetrahydrofuran).

[0104] The following illustrates specific examples of compounds ofstructural formula (I) and synthetic routes to some of these structures.

Preparation of Example 1

[0105]

EXAMPLE 1

[0106] Example 1 was prepared from a tryptophan ester (Intermediate 1).The tryptophan ester utilized to prepare Example 1 is availablecommercially from Aldrich Chemical Co., Milwaukee, Wis. ThePictet-Spengler reaction to provide Example 1 from Intermediate 4 isdisclosed in T. A. Miller et al., Bioorg. Med. Chem. Lett., 8, 1065(1998); J. J. Tepe et al., J. Med. Chem., 39, 2188 (1996); C. W. Ong etal., Aust. J. Chem., 43, 773 (1990); and R. H. Herbert et al.,Tetrahedron Lett., 35, 5497 (1994).

[0107] Preparation of Cbz-Protected Amide Intermediate 2

[0108] Dicyclohexylcarbodiimide (1.8 g, 8.8 mmol) was added to asolution of N-Cbz-N,2-dimethylalanine (2.0 g, 8.0 mmol) in methylenechloride (10 mL) at 0° C. under a nitrogen blanket in one portion. Theresulting white slurry was stirred at 0° C. for 30 min, after which aslurry of Intermediate 1 (1.7 g, 8.0 mmol) and triethylamine (1.3 mL, 10mmol) in methylene chloride (10 mL) was added over 5 min. The resultingwhite slurry was slowly warmed to room temperature, then stirred for atotal of 6 days. The slurry then was filtered under reduced pressure,and the solid was washed with CH₂Cl₂ (50 mL). The filtrate wasconcentrated under reduced pressure to provide a yellow oil, which waspurified by flash column chromatography, eluting with ethylacetate/chloroform (1:9), to provide Cbz-protected amide Intermediate 2as a white semi-solid (3.76 g, 100%): TLC R_(f) (1:4 ethylacetate/chloroform)=0.35; ¹H NMR (300 MHz, DMSO-d₆): δ10.85 (s, 1H),7.76 (d, J=7.6 Hz, 1H), 7.46 (d, J=7.6 Hz, 1H), 7.34-7.26 (m, 5H), 7.12(s, 1H), 7.06-6.94 (m, 2H), 4.88-4.83 (m, 1H), 4.46 (q, J=8.0 Hz, 1H),3.53 (s, 3H), 3.09 (t, J=6.5 Hz, 2H), 2.89 (2, 3H), 1.29 (s, 3H), 1.23(s, 3H) ppm.

[0109] Preparation of Methylamine Intermediate 3

[0110] A solution of Intermediate 2 (3.7 g, 8.2 mmol) in acetic acid (10mL) and methanol (50 mL) was treated with a catalytic amount of 10%palladium on carbon (0.40 g, 50% wet) and the resulting mixture wasstirred under a hydrogen atmosphere at 50° C. for 29 hours. The reactionmixture then was cooled to room temperature, and the palladium catalystwas removed by vacuum filtration through a plug of Celite. The filtratewas concentrated under reduced pressure to provide a yellow residue,which was purified by flash column chromatography, eluting withmethanol/chloroform (1:9), to provide Intermediate 3 as an amber oil(1.65 g, 65%): TLC R_(f) (1:9 methanol/chloroform)=0.55; ¹H NMR (300MHz, CDCl₃): δ8.17 (s, 1H), 7.70 (d, J=7.9 Hz, 1H), 7.59 (d, J=7.8 Hz,2H), 7.35 (d, J=7.7 Hz, 1H), 7.19-7.12 (m, 2H), 4.87 (q, J=5.3 Hz, 1H),3.72 (s, 3H), 3.37-3.30 (m, 2H), 2.27 (s, 3H), 1.38 (s, 3H), 1.33 (s,3H) ppm; API MS m/z 318 [C₁₇H₂₃N₃O₃+H]⁺.

[0111] Preparation of Diketopiperazine Intermediate 4

[0112] A suspension of Intermediate 3 (1.6 g, 5.0 mmol) in ethanol (50mL) was heated to reflux under a nitrogen blanket and stirred for atotal of 3 days. The solvent was removed under reduced pressure toprovide a residue which was purified by flash column chromatography,eluting with methanol/chloroform (1:19), to provide diketopiperazineIntermediate 4 as a white foam (0.810 g, 56%): mp 223-229° C.; TLC R_(f)(1:9 methanol/chloroform)=0.63; ¹H NMR (300 MHz, DMSO-d₆): δ10.86 (s,1H), 8.12 (s, 1H), 7.50 (d, J=7.9 Hz, 1H), 7.28 (d, J=8.0 Hz, 1H),7.01-6.93 (m, 3H), 4.19 (s, 1H), 3.29-3.28 (m, 1H), 2.98 (dd, J=4.6,14.2 Hz, 1H), 2.57 (s, 3H), 1.22 (s, 6H) ppm; CI MS (methane) m/z 286[C₁₆H₁₉N₃O₂+H]⁺; [α]_(D) ^(25° C.)=−26.5° (c=0.10, DMSO).

Preparation of Example 1(12a,R)-6-Benzo[1,3]dioxol-5-yl,2,3,3-trimethyl-2,3,6,7,12,12a-hexahydropyrazino[1′,2′:1,6]-pyrido[3,4-b]indole-1,4-dione

[0113] Trifluoroacetic acid (0.5 mL, 6.2 mmol) was added to a suspensionof Intermediate 4 (0.800 g, 2.8 mmol) and piperonal (0.505 g, 3.4 mmol)in methylene chloride (10 mL) at 0° C. under a nitrogen blanket, afterwhich the solution was warmed to room temperature and stirred for atotal of 22 hours. The reaction mixture was diluted with ethyl acetate(150 mL), washed with saturated sodium bicarbonate (NaHCO₃) solution(100 mL) and brine (50 mL), treated with silica gel (5 g), then thesolvent was removed under reduced pressure. The residue was purified byflash column chromatography, eluting with methanol/chloroform (1:49), toprovide Example 1 as a white powder (0.534 g, 46%, 1:1 mixture of cisand trans diastereomers at C6): mp 225-235° C.; TLC R_(f) (1:19methanol/chloroform)=0.87; ¹H NMR (300 MHz, DMSO-d₆): δ10.08 (s, 2H),8.37 (s, 1H), 8.25 (s, 1H), 7.46 (t, J=8.9 Hz, 2H), 7.28 (t, J=8.2 Hz,2H), 7.02-6.95 (m, 4H), 6.80 (d, J=8.0 Hz, 2H), 6.60 (s, 1H), 6.48 (d,J=8.1 Hz, 1H), 6.15 (s, 1H), 5.98 (d, J=4.4 Hz, 2H), 4.19 (s, 2H), 3.22(dd, J=3.9, 14.8 Hz, 2H), 3.00-2.89 (m, 2H), 2.70 (s, 3H), 2.62 (s, 3H),1.35 (s, 3H), 1.31 (s, 3H), 0.65 (s, 3H), 0.47 (s, 3H) ppm; API MS m/z418 [C₂₄H₂₃N₃O₄+H]⁺; [α]_(D) ^(25° C.)=−63.1° (c=0.50, CHCl₃). Anal.Calcd. for C₂₄H₂₃N₃O₄: C, 69.05; H, 5.55; N, 10.07. Found: C, 68.91; H,5.56; N, 10.04. Chiral HPLC analysis (Chiralcel OD Column, 250×4.6 mm,Retention Time=7.34 min; 1:1 isopropanol/hexanes; flow=0.5 mL/min;detector @ 254 nm; 25° C.) showed one major peak, with a purity of99.9%.

Preparation of Example 2

[0114]

[0115] Example 2 was prepared from the compound of structural formula(III) and N-Cbz-L-glutamine as illustrated below.

[0116] Preparation of cis-β-Carboline Amide Intermediate 6

[0117] To a solution of N-Cbz-L-glutamine (Intermediate 5) (5.0 g, 17.8mmol) in THF (80 mL) at 0° C. under a nitrogen blanket was addeddicyclohexylcarbodiimide (4.4 g, 21.4 mmol) in one portion. Theresulting white slurry was stirred at 0° C. for 30 minutes, after whichCompound (III) (6.3 g, 17.8 mmol) was added in one portion. Theresulting amber slurry was slowly warmed to room temperature, thenstirred 48 hours. The slurry was filtered under reduced pressure and thesolid was washed with methylene chloride (500 mL). The filtrate wasconcentrated under reduced pressure to provide an amber foam, which waspartially purified by flash column chromatography, eluting withmethanol/chloroform (1:9), to provide Intermediate 6 as an off-whitefoam (4.6 g): TLC R_(f) (9:1 methanol/chloroform)=0.60.

Preparation of Example 23-((3S,6R,12aR)-6-Benzo[1,3]dioxol-5-yl-1,4-dioxo-1,2,3,4,6,7,12,12a-octahydropyrazino[1′,2′:1,6]-pyrido[3,4-b]indo-3-yl)propionamide

[0118] A solution of Intermediate 6 (4.6 g, 7.5 mmol) in methanol (50mL) and acetic acid (5 mL) was treated with a catalytic amount of 10%palladium on carbon (500 mg, 50% wet) and the resulting mixture wasstirred under a hydrogen atmosphere at 50° C. for 3 hours. The reactionmixture then was cooled to room temperature and the palladium catalystwas removed by vacuum filtration through Celite (10 g). The filtrate wasconcentrated under reduced pressure and the residue was purified byflash column chromatography, eluting with methanol/chloroform (1:9), toprovide Example 2 as a white powder (1.24 g, 15% over two steps): mp78-82° C.; TLC R_(f) (4:1 chloroform/methanol)=0.58; ¹H NMR (300 MHz,DMSO-d₆): δ11.05 (s, 1H), 8.57 (d, J=3.8 Hz, 1H), 7.52 (d, J=7.3 Hz,1H), 7.30 (d, J=7.7 Hz, 1H), 7.23 (s, 1H), 7.08-6.97 (m, 2H), 6.78 (s,1H), 6.10 (s, 1H), 5.92 (s, 2H), 4.43 (dd, J=4.4, 11.4 Hz, 1H),3.83-3.80 (m, 1H), 3.61 (dd, J=4.5, 15.8 Hz, 1H), 3.32 (s, 3H), 2.93(dd, J=11.7, 15.8 Hz, 1H), 2.17 (t, J=7.2 Hz, 2H), 1.94 (t, J=7.4 Hz,2H) ppm; API MS m/z 447 [C₂₄H₂₂N₄O₅+H]⁺. Anal. Calcd. forC₂₄H₂₂N₄O₅.H₂O: C, 62.06; H, 5.21; N, 12.06. Found: C, 62.12; H, 5.00;N, 12.07. The relative stereochemistry of analog Example 2 was confirmedto be the cis isomer by a series of NOE difference experiments: positiveNOE enhancements from the C12a proton at 4.45 ppm to the C₆ proton at6.10 ppm; a positive NOE enhancement from the C6 proton at 6.10 ppm tothe C12a proton at 4.45 ppm.

Preparation of Example 3

[0119]

[0120] Example 3 was prepared from the hydrochloride of Compound (III)by the following synthetic sequence. The 1,2,4-triazine-3,6-dione ofExample 3 was prepared by the method of K. Winterfield et al., Arch.Pharmaz., 304, 0. 216 (1971).

[0121] Preparation of cis-β-Carboline Carbamate Intermediate 7

[0122] Methyl chloroformate (4.8 mL, 62 mmol) was added dropwise to asuspension of Compound (III) (20 g, 52 mmol) and N-methylmorpholine(14.2 mL, 129 mmol) in THF (150 mL) at 0° C. under a nitrogen blanket.The mixture was slowly warmed to room temperature, then stirred for 3days. The resulting mixture was diluted with ethyl acetate (200 mL),washed with brine (150 mL), dried over magnesium sulfate (MgSO₄), andfiltered. The solvent was removed under reduced pressure to affordIntermediate 7 as an amber foam (21 g, 96%), which was suitable for usewithout further purification.

Preparation of Example 3(5aR,10R)-10-Benzo[1,3]dioxol-5-yl-5,5a,7,8,10,11-hexahydro-7,8,9a,11-tetraazabenzo[b]fluorene-6,9-dione

[0123] Sodium methoxide (5.2 mL, 27 mmol, 30% solution in methanol) wasadded dropwise to a mixture of Intermediate 7 (5 g, 12 mmol) andanhydrous hydrazine (0.5 mL, 15 mmol) in ethanol (30 mL), and themixture was heated at reflux under a nitrogen blanket for 27 hours. Thesuspension was cooled to room temperature and the orange solids wereremoved by vacuum filtration. The organic phase was concentrated underreduced pressure to afford an oxange foam which was purified by reversephase flash column chromatography on C-18 silica gel, elutinq withmethanol/water (1:2), to provide the crude product as an orange oil. Theoil was further purified by flash column chromatography, eluting withmethanol/chloroform (1:4), to provide Example 3 as a yellow powder(0.465 g, 10%): mp 188-194° C.; TLC R_(f) (1:4methanol/chloroform)=0.37; ¹H NMR (300 MHz, D₂O): δ7.72 (d, J=7.6 Hz,1H), 7.42 (t, J=8.6 Hz, 1H), 7.30-7.23 (m, 2H), 7.00 (s, 1H), 6.23-6.83(m, 1H), 6.04 (d, J=11.1 Hz, 2H), 5.73 (s, 1H), 4.18 (dd, J=7.0, 11.5Hz, 1H), 3.51 (dd, J=5.0, 11.4 Hz, 1H), 3.22 (t, J=15.3 Hz, 1H) ppm; APIMS m/z 377 [C₂₀H₁₆N₄O₄+H]⁺; [α]_(D) ^(25° C.)=+44.7° (c=0.5, methanol).Anal. Calcd. for C₂₀H₁₆N₄O₄.0.25 H₂O: C, 63.07; H, 4.37; N, 14.71.Found: C, 63.06; H, 4.30; N, 14,71. The relative stereochemistry ofExample 3 was confirmed to be the cis isomer by a series of NOEdifference experiments: a positive NOE enhancement from the C12a protonat 3.57 ppm to the C6 proton at 5.73 ppm; a positive NOE enhancementfrom the C6 proton at 5.73 ppm to the C12a proton at 3.57 ppm.

Preparation of Example 4

[0124]

[0125] The compound of Example 4 was prepared from Compound (III) by thefollowing reaction scheme.

[0126] Preparation of (+)-cis-3-Chloropropionyl-β-Carboline Intermediate8

[0127] To a mixture of Compound (III) (6.00 g, 15.5 mmol) andtriethylamine (6.5 mL, 46.5 mmol) in THF (100 mL) and water (25 mL), wasadded 3-chloropropionyl chloride (1.9 mL, 20.2 mmol), dropwise, at 0° C.under a nitrogen blanket. The resulting mixture was warmed to roomtemperature and stirred for 1.5 hours. The reaction was quenched with 1N HCl (40 mL) and was concentrated to remove THF. The residue wasdiluted with EtOAc (200 mL), then the layers were separated. The organicphase was washed with 1 N HCl (50 mL), saturated NaHCO₃ (50 mL), andsaturated sodium chloride (NaCI) (50 mL), then dried over anhydrousNa₂SO₄. Filtration and concentration in vacuo gave an oil which waspurified by column chromatography (silica gel, 0-5% EtOAc/CH₂Cl₂). Theproduct was obtained as a pale yellow foam 2.30 g (33.6%): TLC R_(f)(90:10:1/CH₂Cl₂:EtOAc:MeOH)=0.80; ¹H NMR (300 MHz, CDCl): δ10.86 (s,1H), 7.54 (d, J=7.6 Hz, 1H), 7.28 (d, J=7.9 Hz, 1H), 7.10 (t, J=7.4 Hz,1H), 7.02 (t, J=7.3 Hz, 1H), 6.85 (s, 1H), 6.79 (d, J=8.1 Hz, 1H), 6.67(s, 1H), 6.44 (d, J=8.0 Hz, 1H), 6.27 (s, 1H), 5.97 (d, J=4.5 Hz, 2H),5.28 (d, J=6.6 Hz, 1H), 3.90 (t, J=6.5 Hz, 2H), 3.43 (d, J=16.0 Hz, 1H),3.17-3.37 (m, 2H), 2.91-3.06 (m, 4H).

Preparation of Example 4(5aS,11R)-11-Benzo[1,3]dioxol-5-yl-7-methyl-5,5a,8,9,11,12-hexahydro-7H-7,10a,12-triaza-cyclohepta[b]fluorene-6,10-dione

[0128] A mixture of Intermediate 8 (1.22 g, 2.77 mmol), 40% methylaminein water (1.2 mL, 13.8 mmol), and THF (20 mL) was stirred at 45° C.under a nitrogen blanket. After 4 hours, only starting material and the3-methylamino intermediate were detected. Sodium iodide (20.7 mg, 0.138mmol) and 40% methylamine in water (1.2 mL) were added, and the mixturewas stirred at 45° C. for 4 days. The mixture was cooled to roomtemperature and was quenched with 1 N HCl (2 mL). The residue wasdiluted with CH₂Cl₂ (100 mL), washed with 0.1 N HCl (10 mL), water (30mL), and saturated NaCl (20 mL), then dried over anhydrous Na₂SO₄.Filtration and concentration in vacuo gave a yellow foam which aspurified by column chromatography (silica gel,90:10:1/CH₂Cl₂:EtOAc:MeOH). The product was obtained as a white solid0.55 g (49.5%) after recrystallization from MeOH/water. The product wascontaminated with approximately 8% of the cis isomer: mp 179-182° C.;TLC R_(f) (90:10:1/CH₂Cl₂:EtOAc:MeOH)=0.27; ¹H NMR (300 MHz, DMSO-d₆):δ11.08 (s, 1H), 7.48 (d, J=7.7 Hz, 1H), 7.32 (d, J=7.9 Hz, 1H), 7.10 (t,J=7.5 Hz, 1H), 7.01 (t, J=7.2 Hz, 1H), 6.86 (d, J=8.0 Hz, 1H), 6.83 (s,1H), 6.79 (d, J=1.5 Hz, 1H), 6.59 (d, J=8.7 Hz, 1H), 6.01 (d, J=2.3 Hz,2H), 4.45 (dd, J=4.1 Hz, J=11.0 Hz, 1H), 3.74-3.82 (m, 1H), 3.27-3.45(m, 2H), 3.08 (dd, J=4.1 Hz, J=15.4 Hz, 1H), 2.91-3.00 (m, 4H),2.74-2.81 (m, 1H); MS (API) m/z 404 (M+H), 426 (M+Na); [α]_(D)^(25° C.)=−140.7° (c=0.17, DMSO). Anal. Calcd. for C₂₃H₂₁N₃O₄.0.4 H₂O:C, 67.27; H, 5,35; N, 10.23. Found: C, 67.28; H, 5.30; N, 10.09. Therelative stereochemistry of the major product was confirmed to be thetrans isomer by NOE difference experiments (DMSO-d₆): no NOEenhancements from the C5a proton at 1.29 ppm to the C11 proton at 6.83ppm.

Preparation of Example 5

[0129]

[0130] The compound of Example 5 was prepared from Compound (III) and(L)-N-Cbz-γ-tert-butyl-glutamic acid by the following synthetic scheme.

[0131] Preparation of Intermediate 9

[0132] To a solution of L-Cbz-γ-tert-butyl-glutamic acid (5.0 g, 14.8mmol) in methylene chloride (30 mL) at 0° C. was addeddicyclohexylcarbodiimide (3.09 g, 15 mmol) in one portion. The resultingwhite slurry was stirred at 0° C. for 30 minutes, after which Compound(III) was added. The resulting yellow slurry was slowly warmed to roomtemperature, and stirred for a total of 18 hours. The slurry wasfiltered under reduced pressure and the solid was washed with methylenechloride (5×10 mL). The filtrate was concentrated under reduced pressureand the residue was purified by flash column chromatography, elutingwith methylene chloride/ethyl acetate (3:1), to provide the impureIntermediate 9 (containing a small amount of dicyclohexylcarbodiimide)as a yellow solid which was used without further purification (8.3 g):TLC R_(f) (4:1 methylene chloride/ethyl acetate)=0.91.

Preparation of Example 53-((3S,6R,12aR)-6-Benzo[1,3]dioxol-5-yl-1,4-dioxo-1,2,3,4,6,7,12,12a-octahydropyrazino[1′,2′:1,6]-pyrido[3,4-b]indol-3-yl-propionicacid tert-butyl ester

[0133] Intermediate 9 was dissolved in methanol (100 mL) and treatedwith a catalytic amount of 5% palladium on carbon (0.50 g, about 50%wet). The mixture was stirred under a hydrogen atmosphere at 50° C. for6 hours, after which the palladium catalyst was removed by vacuumfiltration through a plug of Celite, eluting with methanol (100 mL). Thefiltrate was concentrated under reduced pressure and the residue waspurified by flash column chromatography, eluting with methylenechloride/ethyl acetate (4:1), to provide Example 5 as a white solid (5.1g, 69% over two steps). A small lot of this product was further purifiedby recrystallization from methylene chloride at −10° C.: mp 222-227° C.;TLC R_(f) (4:1 methylene chloride/ethyl acetate)=0.40; ¹H NMR (300 MHz,CDCl₃): δ7.74 (bs, 1H), 7.65-7.56 (m, 1H), 7.30-7.10 (m, 3H), 6.90-6.83(m, 1H), 6.75-6.67 (m, 2H), 6.26 (d, J=3.3 Hz, 1H), 5.19 (s, 1H), 5.89(s, 1H), 5.86 (s, 1H), 4.41 (dd, J=5.7, 4.1 Hz, 1H), 4.10-4.02 (m, 1H),3.76 (dd, J=8.0, 4.1 Hz, 1H), 3.30-3.17 (m, 1H), 2.42 (t, J=7.1 Hz, 2H),2.28-2.00 (m, 2H), 1.44 (s, 9H); ¹³C NMR (125 MHz, CDCl₃): δ171.8,168.7, 167.9, 147.9, 136;5, 135.6, 132.9, 126.3, 122.6, 120.6, 120.2,118.6, 111.2, 108.3, 107.3, 106.3, 101.2, 81.2, 57.2, 55.4,.55.2, 31.3,29.4, 28.1, 28.8 ppm; API MS m/z 504 [C₂₈H₂₉N₃O₆+H]⁺; [α]_(D)^(25° C.)=+31.3° (c=1.0, CHCl₃). Anal. Calcd. for C₂₈H₂₉N₃O₈: C, 66.79;H, 5.80; N, 8.34. Found: C, 66.27; H, 5.74; N, 8.30. The relativestereochemistry of Example 5 was confirmed to be the cis isomer by anNOE difference experiment (DMSO-d₆): positive NOE enhancements from theC12a proton at 4.40 ppm to the C6 proton at 6.05 ppm (1.3%) and a C12proton at 3.45 ppm (3.3%). HPLC analysis by the previously describedmethod showed one-major peak with a purity of 99.1%.

Preparation of Example 63-((3S,6R,12aR)-6-Benzo[1,3]dioxol-5-yl-1,4-dioxo-1,2,3,4,6,7,12,12a-octahydropyrazino[1′,2′:1,6]pyrido[3,4-b]indole-3-yl)propionicacid

[0134]

[0135] Example 6 was prepared from Example 5 by the following reaction.

EXAMPLE 6

[0136] To a mixture of Example 5 (1.13 g, 2.25 mmol) in acetic acid (11mL) was added concentrated sulfuric acid (0.30 mL) dropwise. Theresulting purple solution was stirred at room temperature for 30minutes, after which water (50 mL) was added. The resulting white slurrywas stirred at room temperature for 30 minutes. The solid was collectedby vacuum filtration, and the solid residue was dissolved in a 70° C.mixture of acetic acid and water (2:1, 50 mL). The yellow solution thenwas cooled to room temperature, then additional water (50 mL) was added.The solid was collected by vacuum filtration, and dried in a vacuum ovenat 70° C. overnight to yield Example 6 as a white solid (188 mg). Thefiltrate then was extracted with ethyl acetate to yield additionalExample 6 (550 mg, total yield 74%): mp 221-231° C.; ¹H NMR (300 MHz,DMSO-d₆): δ11.05 (s, 1H), 8.62 (d, J=3.8 Hz, 1H), 7.52 (d, J=7.4 Hz,1H), 7.29 (d, J=7.4 Hz, 1H), 7.12-6.92 (m, 2H), 6.88 (s, 1H), 6.78 (s,2H), 6.09 (s, 1H), 5.92 (s, 2H), 4.44 (dd, J=5.7, 4.4 Hz, 1H), 3.92-3.79(m, 1H), 3.58-3.42 (m, 1H), 3.02-2.88 (m, 1H), 2.35 (t, J=7.3 Hz, 2H),2.05-1.82 (m, 2H); ¹³C NMR (125 MHz, DMSO-d₆) δ173.7, 168.7, 167.7,147.0, 146.0, 137.2, 136.1, 134.1, 125.7, 121.1, 119.0, 118.8, 118.0,111.2, 108.0, 106.8, 104.5, 100.8, 55.5, 54.6, 54.3, 29.6, 28.4, 22.9ppm; API MS m/z 448 [C₂₁H₂₀N₄O₃+H]⁺; [α]_(D) ^(25° C.)=+69.17° (c=0.5,DMSO). Anal. Calcd. for C₂₁H₂₀N₄O₃.0.75 H₂O: C, 62.64; H, 4.76; N, 9,13.Found: C, 62.55; H, 4.82; N, 9.00.

Preparation of Example 73-((3S,6R,12aR)-6-Benzo[1,3]dioxol-5-yl-1,4-dioxo-1,2,3,4,6,7,12,12a-octahydropyrazino[1′,2′:1,6]pyrido[3,4-b]indol-3-yl)-propionicacid isopropyl ester

[0137]

[0138] Example 7 was prepared from Example 6 by the following reaction.

[0139] To a solution of Example 6 (558 mg, 1.25 mmol) and4-(dimethylamino)pyridine (DMAP, 60 mg, 0.49 mmol) in isopropanol (1.0mL, 13.10 mmol) and methylene chloride (10 mL), at 0° C. under anitrogen blanket, was added dicyclohexylcarbodiimide (DCC, 409 mg, 1.99mmol) in one portion. The resulting white slurry was stirred at roomtemperature for 2 days. The white slurry was filtered under reducedpressure, then the solid was washed with methylene chloride (3×10 mL).The filtrate was concentrated under reduced pressure and the residue waspurified by flash column chromatography, eluting with methylenechloride/ethyl acetate (4:1), to provide Example 7 (450 mg), whichcontained residual dicyclohexylcarbodiimide. This solid was dissolved inmethylene chloride (4 mL), and the solution was stored at −10° C.overnight. The white crystals that formed were quickly filtered througha cold funnel under vacuum. The solid was washed with cold methylenechloride (2×1 mL), then dried in a vacuum oven at 60° C. for 24 hours toprovide Example 7 as a white solid (186 mg, 30%): TLC R_(f) (4:1methylene chloride/ethyl acetate)=0.31; ¹H NMR (300 MHz, DMSO-d₆):δ11.05 (s, 1H), 8.60 (d, J=3.7 Hz, 1H), 7.53 (d, J=7.4 Hz, 1H), 7.30 (d,J=7.4 Hz, 1H), 7.12-6.93 (m, 2H), 6.87 (s, 1H), 6.78 (s, 2H), 6.09 (s,1H), 5.92 (s, 2H), 4.87 (sep, J=6.2 Hz, 1H), 4.44 (dd, J=5.7, 4.4 Hz,1H), 3.90-3.79 (m, 1H), 3.55-3.47 (m, 1H), 3.02-2.87 (m, 1H), 2.45-2.30(m, 2H), 2.03-1.87 (m, 2H), 1.22-1.05 (m, 6H); ¹³C NMR (125 MHz,DMSO-d₆): δ171.6, 168.5, 167.7, 147.0, 146.0, 137.2, 136.1, 134.1,125.7, 121.1, 119.0, 118.8, 118.0, 111.2, 108.0, 106.8, 104.5, 100.8,67.1, 55.6, 54.6, 54.3, 29.8, 28.4, 22.9, 21.6 ppm; API MS m/z 491[C₂₇H₂₇N₃O₆+H]⁺; [α]_(D) ^(25° C.)=+47.3° (c=0.5, CHCl₃). Anal. Calcd.for C₂₇H₂₇N₃O₆: C, 66.25; H, 5.56; N, 8.58. Found: C, 65.85; H, 5.63; N,8.54. The stereochemistry was confirmed to be the cis isomer by an NOEdifference experiment: positive NOE enhancements from the C12a proton at4.44 ppm to the C6 proton at 6.09 ppm (1.8%) and a C12 proton at 3.50ppm (2.7%).

Preparation of Example 8

[0140]

[0141] The compound of Example 8 was prepared from Compound (III) andN-Cbz-(R)-(+)-oxazolidinecarboxylic acid as depicted in the followingsynthetic sequence.

EXAMPLE 8

[0142] Preparation of Intermediate 10

[0143] To a solution of Cbz-(R)-(+)-oxazolidine-carboxylic acid (1.3 g,5.1 mmol) in chloroform (20 mL), at 0° C., was addeddicyclohexylcarbodiimide (1.1 g, 5.3 mmol) in one portion. The resultingwhite slurry was stirred at 0° C. for 30 minutes, after which Compound(III) (1.7 g, 5.0 mmol) was added to the mixture. The resulting yellowslurry was warmed to room temperature, after which the slurry was heatedat 60° C. for 6 days. The solution then was cooled to room temperature,concentrated under reduced pressure to one-third of the slurry volume,and the resulting solid removed by vacuum filtration. The filtrate wasconcentrated under reduced pressure, and the residue was purified byflash column chromatography, eluting with methylene chloride/acetone(1:0 to 32:1), to provide Intermediate 10 as a colorless solid (0.56 g,19%): TLC R_(f) (32:1 methylene chloride/acetone)=0.34.

Preparation of Example 8(3R,6R,12aR)-6-Benzo[1,3]dioxo-5-yl-3-hydroxymethyl-2,3,6,7,12,12a-hexahydropyrazino[1′,2′:1,;6]pyrido[3,4-b]indole-1,4-dione

[0144] Intermediate 9 (0.55 g, 0.95 mmol) in methanol (50 mL) and aceticacid (15 mL) was treated with a catalytic amount of 10% palladium oncarbon (0.08 g, about 10% wet). The mixture was stirred under a hydrogenatmosphere at 55° C. for 3 hours, after which the palladium catalyst wasremoved by vacuum filtration through a plug of Celite, eluting withmethanol. The filtrate was concentrated under reduced pressure toprovide a yellow oil which was purified by flash column chromatography,eluting with methylene chloride/ethyl acetate/methanol (9:1:0.5), toyield Example 8 as a white foamy solid (0.17 g, 44%): mp 194-200° C.;TLC R_(f) (8:1:0.5 methylene chloride/ethyl acetate/methanol)=0.43; ¹HNMR (300 MHz, CDCl₃): δ7.87 (s, 1H), 7.58 (d, J=6.6 Hz, 1H), 7.29-7.25(m, 2H), 7.20-7.12 (m, 2H), 6.82 (m, 1H), 6.73-6.64 (m, 3H), 6.16 (s,1H), 5.86 (d, J=5.7 Hz, 2H), 4.39-4.33 (m, 1H), 4.15-4.12 (m, 1H),4.02-3.90 (m, 2H), 3.75-3.69 (m, 1H), 3.28-3.19 (m, 1H); ¹³C NMR (75MHz, CDCl₃): δ168.7, 148.6, 147.3, 136.7, 135.6, 132.9, 126.5, 122.8,120.4, 120.1, 118.8, 111.4, 108.6, 107.1, 106.4, 101.3, 62.9, 56.9,56.4, 55.6, 23.3 ppm; CI MS (methane) m/z 406 [C₂₂H₁₉N₃O₅+H]⁺; [α]_(D)^(25° C.)=+58.7 (c=0.15, methanol). Anal. Calcd. for C₂₃H₁₉N₃O₅.0.75H₂O: C, 63.08; H, 4.93; N, 10.03. Found: C, 63.17; H, 4.90; N. 9.79.

Preparation of Example 9

[0145]

[0146] The compound of Example 9 can be prepared by the followingsynthetic sequence.

[0147] The following Examples 10-37 were prepared by methods analogousto Examples 1-9.

Example X R² ¹⁾ R¹ 1

1 CH₃ 2

1 H 3

1 H 4

1 CH₃ 5

1 H 6

1 H 7

1 H 8

1 H 9

1 CH₃ 10

1 CH₃ 11

1 H 12

1 H 13

1 H 14

1 CH₃ 15

1 H 16

1 H 17

1 CH₃ 18

1 H 19

1 H 20

1 H 21

1 H 22

1 H 23

1 H 24

1 H 25

1 H 26

1 H 27

1 H 28

1 H 29

1 H 30

1 H 31

1 H 32

1 H 33

1 H 34

1 H 35

1 H 36

1 H 37

1 H

[0148]

[0149] and the designation 2 is

[0150] Compounds of the present invention can be formulated into tabletsfor oral administration. For example, a compound of formula (I) can beformed into a dispersion with a polymeric carrier by the coprecipitationmethod set forth in WO 96/38131, incorporated herein by reference. Thecoprecipitated dispersion then can be blended with excipients, thenpressed into tablets, which optionally are film-coated.

[0151] The compounds of structural formula (I) were tested for anability to inhibit PDE5. The ability of a compound to inhibit PDE5activity is related to the IC₅₀ value for the compound, i.e., theconcentration of inhibitor required for 50% inhibition of enzymeactivity. The IC₅₀ value for compounds of structural formula (I) weredetermined using recombinant human PDE5.

[0152] The compounds of the present invention typically exhibit an IC₅₀value against recombinant human PDE5 of less than about 50 μM, andpreferably less than about 25 μM, and more-preferably less than about 15μm. The compounds of the present invention typically exhibit an IC₅₀value against recombinant human PDE5 of less than about 1 μM, and oftenless than about 0.05 μM. To achieve the full advantage of the presentinvention, a present PDE5 inhibitor has an IC₅₀ of about 0.1 nM to about15 μM.

[0153] The production of recombinant human PDEs and the IC₅₀determinations can be accomplished by well-known methods in the art.Exemplary methods are described as follows:

[0154] Expression of Human PDEs

[0155] Expression in Saccharomyces Cerevisiae (Yeast)

[0156] Recombinant production of human PDE1B, PDE2, PDE4A, PDE4B, PDE4C,PDE4D, PDE5, and PDE7 was carried out similarly to that described inExample 7 of U.S. Pat. No. 5,702,936, incorporated herein by reference,except that the yeast transformation vector employed, which is derivedfrom the basic ADH2 plasmid described in Price et al., Methods inEnzymology, 185, pp. 308-318 (1990), incorporated yeast ADH2 promoterand terminator sequences and the Saccharomyces cerevisiae host was theprotease-deficient strain BJ2-54 deposited on Aug. 31, 1998 with theAmerican Type Culture Collection, Marassas, Va., under accession numberATCC 74465. Transformed host cells were grown in 2× SC-leu medium, pH6.2, with trace metals, and vitamins. After 24 hours,YEP-medium-containing glycerol was added to a final concentration of 2×YET/3% glycerol. Approximately 24 hr later, cells were harvested,washed, and stored at −70° C.

[0157] Human Phosphodiesterase Preparations

[0158] Phosphodiesterase Activity Determinations

[0159] Phosphodiesterase activity of the preparations was determined asfollows. PDE assays utilizing a charcoal separation technique wereperformed essentially as described in Loughney et al. (1996). In thisassay, PDE activity converts [32P]cAMP or [32P]cGMP to the corresponding[32P]5′-AMP or [32P]5′-GMP in proportion to the amount of PDE activitypresent. The [32P]5′-AMP or [32P]5′-GMP then was quantitativelyconverted to free [32P]phosphate and unlabeled adenosine or guanosine bythe action of snake venom 5′-nucleotidase. Hence, the amount of[32P]phosphate liberated is proportional to enzyme activity. The assaywas performed at 30° C. in a 100 μL reaction mixture containing (finalconcentrations) 40 mM Tris HCl (pH 8.0), 1 μM ZnSO₄, 5 mM MgCl₂, and 0.1mg/mL bovine serum albumin (BSA). PDE enzyme was present in quantitiesthat yield <30% total hydrolysis of substrate (linear assay conditions).The assay was initiated by addition of substrate (1 mM [32P]cAMP orcGMP), and the mixture was incubated for 12 minutes. Seventy-five (75)μg of Crotalus atrox venom then was added, and the incubation wascontinued for 3 minutes (15 minutes total). The reaction was stopped byaddition of 200 μL of activated charcoal (25 mg/mL suspension in 0.1 MNaH₂PO₄, pH 4). After centrifugation (750×g for 3 minutes) to sedimentthe charcoal, a sample of the supernatant was taken for,radioactivitydetermination in a scintillation counter and the PDE activity wascalculated.

[0160] Purification of PDE5 From S. cerevisiae

[0161] Cell pellets (29 g) were thawed on ice with an equal volume ofLysis Buffer (25 mM Tris HCl, pH 8, 5 mM MgCl₂, 0.25 mM DTT, 1 mMbenzamidine, and 10 μM ZnSO₄). Cells were lysed in a Microfluidizer®(Microfluidics Corp.) using nitrogen at 20,000 psi. The lysate wascentrifuged and filtered through 0.45 μm disposable filters. Thefiltrate was applied to a 150 mL column of Q SEPHAROSE® FastFlow(Pharmacia). The column was washed with 1.5 volumes of Buffer A (20 mMBis-Tris Propane, pH 6.8, 1 mM MgCl₂, 0.25 mM DTT, 10 μM ZnSO₄) andeluted with a step gradient of 125 mM NaCl in Buffer A followed by alinear gradient of 125-1000 mM NaCl in Buffer A. Active fractions fromthe linear gradient were applied to a 180 mL hydroxyapatite column inBuffer B (20 mM Bis-Tris Propane (pH 6.8), 1 mM MgCl₂, 0.25 mM DTT, 10μM ZnSO₄, and 250 mM KCl). After loading, the column was washed with 2volumes of Buffer B and eluted with a linear gradient of 0-125 mMpotassium phosphate in Buffer B. Active fractions were pooled,precipitated with 60% ammonium sulfate, and resuspended in Buffer C (20mM Bis-Tris Propane, pH 6.8 , 125 mM NaCl, 0.5 mM DTT, and 10 μM ZnSO₄).The pool was applied to a 140 mL column of SEPHACRYL® S-300 HR andeluted with Buffer C. Active fractions were diluted to 50% glycerol andstored at −20° C.

[0162] The resultant preparations were about 85% pure by SDS-PAGE. Thesepreparations had specific activities of about 3 μmol cGMP hydrolyzed perminute per milligram protein.

[0163] Inhibitory Effect on cGMP-PDE

[0164] cGMP-PDE activity of compounds of the present invention wasmeasured using a one-step assay adapted from Wells et al., Biochim.Biophys. Acta, 384, 430 (1975). The reaction medium contained 50 mMTris-HCl, pH 7.5, 5 mM magnesium acetate, 250 μg/ml 5′-Nucleotidase, 1mM EGTA, and 0.15 μM 8-[H³]-cGMP. Unless otherwise indicated, the enzymeused was a human recombinant PDE5 (ICOS Corp., Bothell, Wash.).

[0165] Compounds of the invention were dissolved in DMSO finally presentat 2% in the assay. The incubation time was 30 minutes during which thetotal substrate conversion did not exceed 30%.

[0166] The IC₅₀ values for the compounds examined were determined fromconcentration-response curves typically using concentrations rangingfrom 10 nM to 10 μM. Tests against other PDE enzymes using standardmethodology showed that compounds of the invention are selective for thecGMP-specific PDE enzyme.

[0167] Biological Data

[0168] The compounds according to the present invention were typicallyfound to exhibit an IC₅₀ value of less than 500 nM (i.e., 0.5 μm). An invitro test data for representative compounds of the invention is givenin the following table TABLE 1 In vitro Results Example PDE5 IC₅₀ (nM) 1161.0 2 2.2 3 113.0 4 593.0 5 0.6 6 1.0 7 0.2 8 28.0 12 0.03 15 8.6 169.5 18 9.2 19 20 20 600 21 420 22 80 23 10 24 90 25 80 26 50 27 90 28 1029 30 30 8.6 31 7.8 32 7.0 33 9.0 34 7.5 35 20 36 7.7 37 8.0

[0169] Obviously, many modifications and variations of the invention ashereinbefore set forth can be made without departing from the spirit andscope thereof, and, therefore, only such limitations should be imposedas are indicated by the appended claims.

What is claimed is:
 1. A compound having a formula

wherein R⁰, independently, is selected from the group consisting of haloand C₁₋₆alkyl; R¹ is selected from the group consisting of hydrogen,C₁₋₆alkyl, C₂₋₆alkenyl, C₂₋₆alkynyl, haloC₁₋₆-alkyl, C₃₋₈cycloalkyl,C₃₋₈cycloalkylC₁₋₃alkyl, arylC₁₋₃-alkyl, and heteroarylC₁₋₃alkyl; R² isselected from the group consisting of an optionally substitutedmonocyclic aromatic ring selected from the group consisting of benzene,thiophene, furan, and pyridine, and an optionally substituted bicyclicring

 wherein the fused ring A is a 5- or 6-membered ring, saturated orpartially or fully unsaturated, and comprises carbon atoms andoptionally one or two heteroatoms selected from oxygen, sulfur, andnitrogen; X is selected from the group consisting of NR^(a), O, S,CR³R⁴, CH₂CR³R⁵, and CR³R⁵CH₂; R³ is selected from the group consistingof hydrogen and C₁₋₃alkyl; or R¹ and R³ together represent a 3- or4-membered alkyl or alkenyl chain component of a 5- or 6-membered ring,or NR^(a), R¹, and the nitrogen to which R¹ is attached, form a 5- or6-membered heterocyclic ring; R⁴ is selected from the group consistingof C₁₋₆alkyl, haloC₁₋₄alkyl, aryl, heteroaryl, Het, C₃₋₈-cycloalkyl,OR^(a), C(═O)OR^(a), C(═O)R^(a), C(═O)NR^(a)SO₂R^(b), C(═O)NR^(a)R^(b),C(═S)NR^(a)R^(b), C₁₋₆alkyleneC(═O)OR^(a), C₁₋₄alkyleneC(═O)NR^(a)R^(b),C₁₋₄alkyleneNR^(a)R^(b), C₁₋₄alkyleneOR^(a),C₁₋₄-alkyleneSO₂NR^(a)R^(b), C₁₋₄alkylenearyl, C₁₋₄alkyleneheteroaryl,C₁₋₄alkyleneHet, C₁₋₄alkyleneC(═O)C₁₋₄alkyleneHet, C₁₋₄alkyleneC(═O)Het,C₁₋₄alkyleneC(═O)OR^(a), C(═O)—C₁₋₄alkyleneHet,C(═O)NR^(a)alkyleneOR^(b), C(═O)NR^(a)C₁₋₄alkyleneHet,NR^(a)C₁₋₄alkyleneNR^(a)R^(b), NR^(a)C(═O)R^(b), NR^(a)C(═O)NR^(a)R^(b),N(SO₂C₁₋₄alkyl)₂, and NR^(a)(SO₂C₁₋₄alkyl), with the proviso that whenR³ is hydrogen, R⁴ is different from C₁₋₃alkyl; or R¹ and R⁴ togetherrepresent a 3- or 4-membered carbocyclic or heterocyclic chaincomponent, either saturated or unsaturated, of a 5- or 6-membered ring;or R³ and R⁴ together represent a 4- to 6-membered alkyl or alkenylchain component of a 5- to 7-membered ring; R⁵ is selected from thegroup consisting of hydrogen, C₁₋₆alkyl, haloC₁₋₄alkyl, aryl,heteroaryl, Het, C₃₋₈cycloalkyl, OR^(a), C(═O)OR^(a), C(═O)R^(a),C(═O)—NR^(a)SO₂R^(b), C(═O)NR^(a)R^(b), C(═S)NR^(a)R^(b),C₁₋₆alkyleneC(═O)OR^(a), C₁₋₄alkyleneC(═O)NR^(a)R^(b),C₁₋₄alkyleneNR_(a)R^(b), C₁₋₄alkylene-OR^(a), C₁₋₄alyleneSO₂NR^(a)R^(b),C₁₋₄alkylenearyl, C₁₋₄alkyleneheteroaryl, C₁₋₄alkyleneHet,C₁₋₄alkyleneC(═O)C₁₋₄-alkyleneHet, C₁₋₄alkyleneC(═O)Het,C₁₋₄alkyleneC(═O)—OR^(a), C(═O)C₁₋₄alkyleneHet,C(═O)NR^(a)alkyleneOR^(b), C(═O)—NR^(a)C₁₋₄alkyleneHet,NR^(a)C₁₋₄alkyleneNR^(a)R^(b), NR^(a)C(═O)R^(b), NR^(a)C(═O)NR^(a)R^(b),N(SO₂C₁₋₄alkyl)₂, and NR^(a)(SO₂C₁₋₄alkyl); R^(a) and R^(b),independently, are selected from the group consisting of hydrogen,C₁₋₈alkyl, aryl, heteroaryl, haloC₁₋₆alkyl, C₁₋₄alkyleneHet,arylC₁₋₃-alkyl, heteroarylC₁₋₃alkyl, C₁₋₄alkylenearyl, C₃₋₈-cycloalkyl,C₁₋₃alkyleneheteroaryl, and Het; Het is a 4- to 7-membered heterocyclicroup, saturated or partially unsaturated, containing at least oneheteroatom selected from the group consisting of oxygen, nitrogen, andsulfur, and optionally substituted with one or more C₁₋₆alkyl,NR^(a)R^(b), and C(═O)OR^(a), q is 0, 1, 2, 3, or 4; andpharmaceutically acceptable salts and solvates thereof.
 2. The compoundof claim 1 represented by the formula

and pharmaceutically acceptable salts and solvates thereof.
 3. Thecompound of claim 1 wherein R⁴ and R⁵ are selected from the groupconsisting of C₁₋₆alkyl, C₁₋₆alkyleneC(═O)OR^(a), C(═O)OR^(a),C(═S)NR^(a)R^(b), C₅₋₇spiro when combined with R³,C₁₋₄alkyleneC(═O)NR^(a)R^(b), C₁₋₄alkyleneNR^(a)R^(b),C₁₋₄alkyleneOR^(a), C₁₋₄alkyleneSO₂NR^(a)R^(b), C₁₋₄alkyleneheteroaryl,haloC₁₋₄alkyl, C₁₋₄alkyleneHet, C₁₋₄alkyleneC(═O)C₁₋₄alkyleneHet, andC₁₋₄alkylenearyl; and R^(a) and R^(b), independently, are selected fromthe group consisting of C₁₋₈alkyl, hydrogen, C₁₋₄alkylenearyl, Het,C₁₋₄alkyleneHet, C₃₋₈cycloalkyl, and haloC₁₋₆alkyl.
 4. The-compound ofclaim 3 wherein Het, aryl, and heteroaryl are optionally substitutedwith one or more of C(═O)OR^(a), NR^(a)R^(b), and C₁₋₆alkyl.
 5. Thecompound of claim 1 wherein X is selected from the group consisting ofCH₂CH₂, NH, and NCH₃.
 6. The compound of claim 1 wherein R³ is selectedfrom the group consisting of hydrogen and methyl.
 7. The compound ofclaim 1 wherein R⁴ is selected from the group consisting of C₁₋₄alkyl,C₁₋₄alkyleneC(═O)NH₂, C₁₋₄alkyleneNH₂, C₁₋₄alkyleneC(═O)—OC₁₋₇alkyl,C₁₋₆alkyleneC(═O)OR^(a), C₁₋₃alkyleneOH, C₁₋₄-alkyleneSO₂NH₂,C₁₋₄alkyleneOC₁₋₃alkylenearyl, haloC₁₋₃alkyl, C₁₋₃alkylenearyl,C₁₋₄alkyleneC(═O)OC₁₋₃haloalkyl,


8. The compound of claim 1 wherein R³ and R⁴ are taken together to forma 5- or 6-membered spiro group.
 9. The compound of claim 1 wherein R² is

wherein n is an integer 1 or 2, and Y and Z, independently, are CH₂, O,S, or NR^(a).
 10. The compound of claim 1 wherein R², substituted orunsubstituted, is selected from the group consisting of


11. A compound of claim 1 selected from the group consisting of

and pharmaceutically acceptable salts and solvates thereof.
 12. Acompound of claim 1 selected from the group consisting of(3S,6R,12aR)-3-(4-aminobutyl)-6-benzo[1,3]dioxol-5-yl-2,3,6,7,12,12a-hexahydropyrazino[1′,2′:1,6]-pyrido[3,4-b]indole-1,4-dione,((3S,6R,12aR)-6-benzo[1,3]dioxol-5-yl-1,4-dioxo-1,2,3,4,6,7,12,12a-octahydropyrazino[1′,2′:1,6]-pyrido[3,4-b]indol-3-yl)aceticacid tert-butyl ester,(3S,6R,12aR)-6-benzo[1,3]dioxol-5-yl-3-benzyloxy-methyl-2,3,6,7,12,12a-hexahydropyrazino[1′,2′:1,6]-pyrido[3,4-b]indole-1,4-dione,((3S,6R,12aR)-6-benzo[1,3]dioxol-5-yl-1,4-dioxo-1,2,3,4,6,7,12,12a-octahydropyrazino[1′,2′:1,6]-pyrido[3,4-b]indol-3-yl)aceticacid,((3R,6R,12aR)-6-benzo[1,3]dioxol-5-yl-1,4-dioxo-1,2,3,4,6,7,12,12a-octahydropyrazino[1′,2′:1,6]-pyrido[3,4-b]indol-3-yl)aceticacid,(6R,12aR)-6-benzo[1,3]dioxol-5-yl-3-pyrazol-1-ylmethyl-2,3,6,7,12,12a-hexahydropyrazino-[1′,2′:1,6]pyrido[3,4-b]indole-1,4-dione,(3S,6R,12aR)-3-(2-aminoethyl)-6-benzo[1,3]dioxol-5-yl-2,3,6,7,12,12a-hexahydropyrazino[1′,2′:1,6]-pyrido[3,4-b]indole-1,4-dione,(3S,6R,12aR)-3-aminomethyl-6-benzo[1,3]dioxol-5-yl-2,3,6,7,12,12a-hexahydropyrazino[1′,2′:1,6]-pyrido[3,4-b]indole-1,4-dione,(3R,6R,12aR)-6-benzo[1,3]dioxol-5-yl-3-chloromethyl-2,3,6,7,12,12a-hexahydropyrazino[1′,2′:1,6]-pyrido[3,4-b]indole-1,4-dione2-((3S,6R,12aR)-6-benzo[1,3]dioxol-5-yl-1,4-dioxo-1,2,3,4,6,7,12,12a-octahydropyrazino[1′,2′:1,6]-pyrido[3,4-b]indol-3-yl)-N-(4-dimethylaminobenzyl)-acetamide,(3S,6R,12aR)-6-benzo[1,3]dioxol-5-yl-3-[2-(4-methyl-piperazin-1-yl)-2-oxo-ethyl]-2,3,6,7,12,12a-hexahydropyrazino[1′,2′:1,6]pyrido[3,4-b]indole-1,4-dione,2-((6R,12aR)-6-benzo[1,3]dioxol-5-yl-1,4-dioxo-1,2,3,4,6,7,12,12a-oxtahydropyrazino[1′,2′:1,6]-pyrido[3,4-b]indol-3-yl)-N-(2-pyrrolidin-1-yl-ethyl)acetamide,(3S,6R,12aR)-6-benzo[1,3]dioxol-5-yl-3-pyridin--3-ylmethyl-2,3,6,7,12,12a-hexahydropyrazino-[1′,2′:1,6]pyrido[3,4-b]indole-1,4-dione,((3S,6R,12aR)-6-benzo[1,3]dioxol-5-yl-1,4-dioxo-1,2,3,4,6,7,12,12a-octahydropyrazino[1′,2′:1,6]-pyrido[3,4-b]indol-3-yl)aceticacid methyl ester,((3S,6R,12aR)-6-benzo[1,3]dioxol-5-yl-1,4-dioxo-1,2,3,4,6,7,12,12a-octahydropyrazino[1′,2′:1,6]-pyrido[3,4-b]indol-3-yl)aceticacid heptyl ester,((3S,6R,12aR)-6-benzo[1,3]dioxol-5-yl-1,4-dioxo-1,2,3,4,6,7,12,12a-octahydropyrazino[1′,2′:1,6]-pyrido[3,4-b]indol-3-yl)aceticacid ethyl ester,((3S,6R,12aR)-6-benzo[1,3]dioxol-5-yl-1,4-dioxo-1,2,3,4,6,7,12,12a-octahydropyrazino[1′,2′:1,6]-pyrido[3,4-b]indol-3-yl)aceticacid isopropyl ester,((3S,6R,12aR)-6-benzo[1,3]dioxol-5-yl-1,4-dioxo-1,2,3,4,6,7,12,12a-octahydropyrazino[1′,2′:1,6]-pyrido[3,4-b]indol-3-yl)aceticacid cyclopentyl ester, ((3S,6R,12aR)-6-benzo[1,3]dioxol-5-yl-1,4-dioxo-1,2,3,4,6,7,12,12a-octahydropyrazino[1′,2′:1,6]-pyrido[3,4-b]indol-3-yl)aceticacid 2,2,2-trifluoroethyl ester,3-((3S,6R,12aR)-6-benzofuran-5-yl-1,4-dioxo-1,2,3,4,6,7,12,12a-octahydropyrazino[1′,2′:1,6]-pyrido[3,4-b]indol-3-yl)propionicacid tert-butyl ester,((3S,6R,12aR)-6-benzofuran-5-yl-1,4-dioxo-1,2,3,4,6,7,12,12a-octahydropyrazino[1′,2′:1,6]-pyrido[3,4-b]indol-3-yl)aceticacid tert-butyl ester,3-((3S,6R,12aR)-6-benzo[1,3]dioxol-5-yl-1,4-dioxo-1,2,3,4,6,7,12,12a-octahydropyrazino[1′,2′:1,6]-pyrido[3,4-b]indol-3-yl)propionicacid ethyl ester, and pharmaceutically acceptable salts and solvatesthereof.
 13. A compound having a formula

wherein R¹ is hydrogen or methyl, R² is

X is selected from the group consisting of


14. A pharmaceutical composition comprising a compound of claim 1,together with a pharmaceutically acceptable diluent or carrier.
 15. Amethod of treating a male or female animal in the treatment of acondition where inhibition of a cGMP-specific PDE is of a therapeuticbenefit comprising treating said animal with an effective amount of apharmaceutical composition comprising a compound of claim 1, togetherwith a pharmaceutically acceptable diluent or carrier.
 16. The method ofclaim 15 wherein the condition is male erectile dysfunction.
 17. Themethod of claim 16 wherein the treatment is an oral treatment.
 18. Themethod of claim 15 wherein the condition is female arousal disorder. 19.The method of claim 18 wherein the treatment is an oral treatment. 20.The method of claim 15 wherein the condition is selected from the groupconsisting of stable angina, unstable angina, variant angina,hypertension, pulmonary hypertension, chronic obstructive pulmonarydisease, malignant hypertension, pheochromocytoma, acute respiratorydistress syndrome, congestive heart failure, acute renal failure,chronic renal failure, atherosclerosis, a condition of reduced bloodvessel patency, a peripheral vascular disease, a vascular disorder,thrombocythemia, an inflammatory disease, myocardial infarction, stroke,bronchitis, chronic asthma, allergic asthma, allergic rhinitis,glaucoma, peptic ulcer, a gut motility disorder, postpercutaneoustransluminal coronary angioplasty, carotid angioplasty, post-bypasssurgery graft stenosis, osteoporosis, preterm labor, benign prostatichypertrophy, and irritable bowel syndrome.
 21. A method of treating acondition where inhibition of a cGMP-specific PDE is of therapeuticbenefit, in a human or a nonhuman animal body, comprising administeringto said body a therapeutically effective amount of a compound ofclaim
 1. 22. A method for the curative or prophylactic treatment of maleerectile dysfunction or female arousal disorder, comprisingadministration of an effective dose of a compound of claim 1, andpharmaceutically acceptable salts and solvates thereof, to an animal.23. Use of a compound of claim 1 for the manufacture of a medicament forthe curative or prophylactic treatment of a condition where inhibitionof a cGMP-specific PDE is of a therapeutic benefit.